Mesocosm study of PAC-modified clay effects on Karenia brevis cells and toxins, chemical dynamics, and benthic invertebrate physiology.

Modified clay compounds are used globally as a method of controlling harmful algal blooms, and their use is currently under consideration to control Karenia brevis blooms in Florida, USA. In 1400 L mesocosm tanks, chemical dynamics and lethal and sublethal impacts of MC II, a polyaluminum chloride (PAC)-modified kaolinite clay, were evaluated over 72 h on a benthic community representative of Sarasota Bay, which included blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). In this experiment, MC II was dosed at 0.2 g L-1 to treat bloom-level densities of K. brevis at 1 × 106 cells L-1. Cell removal in MC II-treated tanks was 57% after 8 h and 95% after 48 h. In the water column, brevetoxin analogs BTx-1 and BTx-2 were found to be significantly higher in untreated tanks at 24 and 48 h, while in MC II-treated tanks, BTx-3 was found to be higher at 48 h and BTx-B5 was found to be higher at 24 and 48 h. In MC II floc, we found no significant differences in BTx-1 or BTx-2 between treatments for any time point, while BTx-3 was found to be significantly higher in the MC II-treated tanks at 48 and 72 h, and BTx-B5 was higher in MC II-treated tanks at 24 and 72 h. Among various chemical dynamics observed, it was notable that dissolved phosphorus was consistently significantly lower in MC II tanks after 2 h, and that turbidity in MC II tanks returned to control levels 48 h after treatment. Dissolved inorganic carbon and total seawater alkalinity were significantly reduced in MC II tanks, and partial pressure of CO2 (pCO2) was significantly higher in the MC II-only treatment after 2 h. In MC II floc, particulate phosphorus was found to be significantly higher in MC II tanks after 24 h. In animals, lethal and sublethal responses to MC II-treated K. brevis did not differ from untreated K. brevis for either of our three species at any time point, suggesting MC II treatment at this dosage has negligible impacts to these species within 72 h of exposure. These results appear promising in terms of the environmental safety of MC II as a potential bloom control option, and we recommend scaling up MC II experiments to field trials in order to gain deeper understanding of MC II performance and dynamics in natural waters.

Discovery of a series of portimine-A fatty acid esters in mussels.

Vulcanodinium rugosum is a benthic dinoflagellate known for producing pinnatoxins, pteriatoxins, portimines and kabirimine. In this study, we aimed to identify unknown analogs of these emerging toxins in mussels collected in the Ingril lagoon, France. First, untargeted data acquisitions were conducted by means of liquid chromatography coupled to hybrid quadrupole-orbitrap mass spectrometry. Data processing involved a molecular networking approach, and a workflow dedicated to the identification of biotransformed metabolites. Additionally, targeted analyses by liquid chromatography coupled to triple quadrupole mass spectrometry were also implemented to further investigate and confirm the identification of new compounds. For the first time, a series of 13-O-acyl esters of portimine-A (n = 13) were identified, with fatty acid chains ranging between C12:0 and C22:6. The profile was dominated by the palmitic acid conjugation. This discovery was supported by fractionation experiments combined with the implementation of a hydrolysis reaction, providing further evidence of the metabolite identities. Furthermore, several analogs were semi-synthesized, definitively confirming the discovery of these metabolization products. A new analog of pinnatoxin, with a molecular formula of C42H65NO9, was also identified across the year 2018, with the highest concentration observed in August (4.5 µg/kg). The MS/MS data collected for this compound exhibited strong structural similarities with PnTX-A and PnTX-G, likely indicating a substituent C2H5O2 in the side chain at C33. The discovery of these new analogs will contribute to deeper knowledge of the chemodiversity of toxins produced by V. rugosum or resulting from shellfish metabolism, thereby improving our ability to characterize the risks associated with these emerging toxins.

Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean.

Dinoflagellates of the genus Gambierdiscus have been associated with ciguatera, the most common non-bacterial fish-related intoxication in the world. Many studies report the presence of potentially toxic Gambierdiscus species along the Atlantic coasts including G. australes, G. silvae and G. excentricus. Estimates of their toxicity, as determined by bio-assays, vary substantially, both between species and strains of the same species. Therefore, there is a need for additional knowledge on the metabolite production of Gambierdiscus species and their variation to better understand species differences. Using liquid chromatography coupled to mass spectrometry, toxin and metabolomic profiles of five species of Gambierdiscus found in the Atlantic Ocean were reported. In addition, a molecular network was constructed aiming at annotating the metabolomes. Results demonstrated that G. excentricus could be discriminated from the other species based solely on the presence of MTX4 and sulfo-gambierones and that the variation in toxin content for a single strain could be up to a factor of two due to different culture conditions between laboratories. While untargeted analyses highlighted a higher variability at the metabolome level, signal correction was applied and supervised multivariate statistics performed on the untargeted data set permitted the selection of 567 features potentially useful as biomarkers for the distinction of G. excentricus, G. caribaeus, G. carolinianus, G. silvae and G. belizeanus. Further studies will be required to validate the use of these biomarkers in discriminating Gambierdiscus species. The study also provided an overview about 17 compound classes present in Gambierdiscus, however, significant improvements in annotation are still required to reach a more comprehensive knowledge of Gambierdiscus' metabolome.

New azaspiracid analogues detected as bi-charged ions in Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters.

Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures.

Paralytic shellfish toxins producing dinoflagellates cause dysbacteriosis in scallop gut microbial biofilms.

Filter-feeding bivalves could accumulate paralytic shellfish toxins (PSTs) produced by harmful dinoflagellates through diet. Despite that bivalves are resistant to these neurotoxins due to possessing PST-resistant sodium channel, exposure to PSTs-producing dinoflagellates impair bivalve survival. We hypothesized that ingesting PSTs-producing dinoflagellates may influence the gut microbiota, and then the health of bivalves. To test this idea, we compared the gut microbiota of the scallop Patinopecten yessoensis, after feeding with PST-producing or non-toxic dinoflagellates. Exposure to PSTs-producing dinoflagellates resulted in a decline of gut microbial diversity and a disturbance of community structure, accompanied by a significant increase in the abundance and richness of pathogenic bacteria, represented by Vibrio. Moreover, network analysis demonstrated extensive positive correlations between pathogenic bacteria abundances and PSTs concentrations in the digestive glands of the scallops. Furthermore, isolation of a dominant Vibrio strain and its genomic analysis revealed a variety of virulence factors, including the tolC outer membrane exporter, which were expressed in the gut microbiota. Finally, the infection experiment demonstrated scallop mortality caused by the isolated Vibrio strain; further, the pathogenicity of this Vibrio strain was attenuated by a mutation in the tolC gene. Together, these findings demonstrated that the PSTs may affect gut microbiota via direct and taxa-specific interactions with opportunistic pathogens, which proliferate after transition from seawater to the gut environment. The present study has revealed novel mechanisms towards deciphering the puzzles in environmental disturbances-caused death of an important aquaculture species.

Toxic responses of metabolites produced by Ostreopsis cf. ovata on a panel of cell types.

Blooms of the dinoflagellate Ostreopsis cf. ovata are regularly associated with human intoxications that are attributed to ovatoxins (OVTXs), the main toxic compounds produced by this organism and close analogs to palytoxin (PlTX). Unlike for PlTX, information on OVTXs'toxicity are scarce due to the absence of commercial standards. Extracts from two cultures of Mediterranean strains of O. cf. ovata (MCCV54 and MCCV55), two fractions containing or not OVTXs (prepared from the MCCV54 extract) and OVTX-a and -d (isolated from the MCCV55 extract) were generated. These chemical samples and PlTX were tested on a panel of cell types from several organs and tissues (skin, intestine, lung, liver and nervous system). The MCCV55 extract, containing a 2-fold higher amount of OVTXs than MCCV54 extract, was shown to be more cytotoxic on all the cell lines and more prone to increase interleukin-8 (IL-8) release in keratinocytes. The fraction containing OVTXs was also cytotoxic on the cell lines tested but induced IL-8 release only in liver cells. Unexpectedly, the cell lines tested showed the same sensitivity to the fraction that does not contain OVTXs. With this fraction, a pro-inflammatory effect was shown both in lung and liver cells. The level of cytotoxicity was similar for OVTX-a and -d, except on intestinal and skin cells where a weak difference of toxicity was observed. Among the 3 toxins, only PlTX induced a pro-inflammatory effect mostly on keratinocytes. These results suggest that the ubiquitous Na+/K+ ATPase target of PlTX is likely shared with OVTX-a and -d, although the differences in pro-inflammatory effect must be explained by other mechanisms.

Advances Toward Amphidinolides C, F and U: Isolations, Synthetic Studies and Total Syntheses.

Amphidinolides C, F, and U, including C2-C4 analogs, are highly cytotoxic marine macrolides, mainly isolated from dinoflagellates of the genus Amphidinium. All these polyketides share a 75 % or more similar structure, highlighted by a macrolactone ring, at least one trans-2,5-substituted-THF motif and a characteristic polyenic side chain. From their isolation and absolute configurational assignment, the total synthesis of these marine macrolides represented an intense challenge to the organic synthesis community over the last 15 years, with around 14 research groups engaged in this inspiring task. In the first part of this review, we present the different approaches to the isolation and characterization of these natural products, including the most recent analogs, which may cast doubt on the biogenetic origin of these compounds. The various synthetic approaches to the total synthesis of C, F, and U amphidinolides are presented in a second part, focusing on key reactions and/or innovative strategies. The review concludes in a third section summarizing the successful approaches leading to the total synthesis of one of the members of this amphidinolide subfamily.

Multispecies mass mortality in the Beagle Channel associated with paralytic shellfish toxins.

The Beagle Channel is a Subantarctic semi-estuarine environment at the southern tip of South America, where intoxication events associated with harmful algal blooms have been reported since 1886, including a world record in toxicity due to Alexandrium catenella in 1992. Toxic algae affect public health and ecosystem services, particularly mussel aquaculture and fisheries management. During the austral summer of 2022, an intense bloom of A. catenella (5 × 104 cells L-1) occurred in the Beagle Channel, leading to the second most toxic event in the area, with mussel toxicity reaching 197,266 µg STXeq kg-1. This event was synchronous with the mortality of marine organisms from different trophic levels and terrestrial fauna, i.e., two Fuegian red foxes and a southern caracara. Stomach content and liver samples from dead kelp gulls (Larus dominicanus), Magellanic penguins (Spheniscus magellanicus), papua penguins (Pygoscelis papua), and imperial cormorants (Leucocarbo atriceps), presented variable paralytic shellfish toxins (PST) levels (up to 3427 µg STXeq kg-1) as measured by high performance liquid chromatography (HPLC), suggesting that deaths were associated with high PST toxicity level. The different toxin profiles found in phytoplankton, zooplankton, squat lobsters (Grimothea gregaria), Fuegian sprat (Sprattus fuegensis), and seabirds evidenced possible toxin transformation along the food web and the possible transfer vectors. The unexpected detection of PST in terrestrial fauna (up to 2707 µg STXeq kg-1) suggested intoxication by scavenging on squat lobsters, which had high toxicity (26,663 µg STXeq kg-1). PST trace levels were also detected in a liver sample of a dead false killer whale (Pseudorca crassidens), an oceanic odontocete stranded on the coast during the bloom. Overall, our results denote the exceptional nature of the toxic, multispecies mortality event and that toxins may propagate to several levels of the food web in this Subantarctic environment.

3-Epimers of Caribbean ciguatoxins in fish and algae.

Ciguatera poisoning (CP) is endemic to several subtropical and tropical regions and is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The recent discovery of Caribbean CTXs (C-CTXs) in Gambierdiscus spp. isolated from the Caribbean resulted in the identification of a precursor analogue, C-CTX5, that is reduced into C-CTX1. C-CTX5 has two reducible sites, a ketone at C-3 and hemiketal at C-56. Chemical reductions of C-CTX5 into C-CTX3/4 resulted in two peaks in the LC-HRMS chromatograms with a ratio that differed markedly from that observed in fish extracts and the reduction of C-CTX1 isolated from fish. Reduction of C-CTX5 should have produced four diastereoisomers of C-CTX3/4, prompting a more detailed study of the reduction products. LC-HRMS with a slow gradient was used to separate and detect the four stereoisomers of C-CTX3/4, and to determine the distribution of these analogues in naturally contaminated fish tissues and following chemical reduction of isolated analogues. The results showed that in naturally contaminated fish tissues C-CTX1/2 is a mixture of two diastereoisomers at C-3 and that C-CTX3/4 is a mixture of two pairs of diastereoisomers at C-3 and C-56. The data suggests that there is variability in the enzymatic reduction at C-3 and C-56 of C-CTXs in reef fish, leading to variations in the ratios of the four stereoisomers. Based on these findings, a naming convention for C-CTXs is proposed which aligns with that used for Pacific CTX congeners and will aid in the identification of the structure and stereochemistry of the different CTX analogues.

Risk characteristics of shellfish toxins in Mytilus unguiculatus around the Zhoushan Islands, East China Sea.

The Zhoushan Islands, are an important area for Mytilus unguiculatus aquaculture, and are threatened by potentially harmful algal blooms. However, a full understanding of the risks posed by their toxin residues is still lacking. M. unguiculatus samples were collected from the area between 2020 and 2021 and analyzed for their toxin profiles to assess the contamination status of shellfish toxins. The main toxins detected were the paralytic shellfish toxins (PSTs), gymnodimine (GYM), and domoic acid (DA). Nine PSTs components were detected, the dominant ones being C1, C2, and GTX5, with an overall detection rate of 85.7 %. The detection rate of DA was 55.05 %, and GYM was detected in all samples. The toxin levels in the samples were significantly lower than the European Union regulatory limits, but toxin contamination was generally universal.

Diversity and distribution of benthic dinoflagellates in Tonga include the potentially harmful genera Gambierdiscus and Fukuyoa.

Benthic dinoflagellates that can cause illness, such as ciguatera poisoning (CP), are prevalent around the Pacific but are poorly described in many locations. This study represents the first ecological assessment of benthic harmful algae species in the Kingdom of Tonga, a country where CP occurs regularly. Surveys were conducted in June 2016 in the Tongatapu island group, and in June 2017 across three island groups: Ha'apai, Vava'u, and Tongatapu. Shallow subtidal coastal habitats were investigated by measuring water quality parameters and conducting quadrat surveys. Microalgae samples were collected using either macrophyte collection or the artificial substrate method. Benthic dinoflagellates (Gambierdiscus and/or Fukuyoa, Ostreopsis, and Prorocentrum) were counted using light microscopy, followed by molecular analyses (real-time PCR in 2016 and high throughput sequencing (metabarcoding) in 2017) to identify Gambierdiscus and Fukuyoa to species level. Six species were detected from the Tongatapu island group in 2016 (G. australes, G. carpenteri, G. honu, G. pacificus, F. paulensis, and F. ruetzleri) using real-time PCR. Using the metabarcoding approach in 2017, a total of eight species (G. australes, G. carpenteri, G. honu, G. pacificus, G. cheloniae, G. lewisii, G. polynesiensis, and F. yasumotoi) were detected. Species were detected in mixed assemblages of up to six species, with G. pacificus and G. carpenteri being the most frequently observed. Ha'apai had the highest diversity with eight species detected, which identifies this area as a Gambierdiscus diversity 'hotspot'. Vava'u and Tongatapu had three and six species found respectively. Gambierdiscus polynesiensis, a described ciguatoxin producer and proposed causative agent of CP was found only in Ha'apai and Vava'u in 2017, but not in Tongatapu in either year. Ostreopsis spp. and Prorocentrum spp. were also frequently observed, with Prorocentrum most abundant at the majority of sites. In 2016, the highest number of Gambierdiscus and/or Fukuyoa cells were observed on seagrass (Halodule uninervis) from Sopu, Tongatapu. In 2017, the highest numbers of Gambierdiscus and/or Fukuyoa from artificial substrate samples were recorded in the Halimeda dominant habitat at Neiafu Tahi, Vava'u, a low energy site. This raised the question of the effect of wave motion or currents on abundance measurements from artificial substrates. Differences in detection were noticed between macrophytes and artificial substrates, with higher numbers of species found on artificial substrates. This study provides a baseline of benthic dinoflagellate distributions and diversity for Tonga that may be used for future studies and the development of monitoring programmes.

Seasonal occurrence of toxic phytoplankton and phycotoxins at a mussel aquaculture site in Nova Scotia, Canada.

A three-year field study at a mussel (Mytilus edulis) aquaculture site in Ship Harbour, Nova Scotia, Canada was carried out between 2004 and 2006 to detect toxic phytoplankton species and dissolved lipophilic phycotoxins and domoic acid. A combination of plankton monitoring and solid phase adsorption toxin tracking (SPATT) techniques were used. Net tow and pipe phytoplankton samples were taken weekly to determine the abundance of potentially toxic species and SPATT samplers were deployed weekly for phycotoxin analysis. Mussels were also collected for toxin analysis in 2005. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse the samples for spirolides (SPXs), pectenotoxins (PTXs), okadaic acid group toxins (OA, DTXs) and domoic acid (DA). Phycotoxins were detected with SPATT samplers beginning from the time of deployment until after the producing organisms were no longer observed in pipe samples. Seasonal changes in toxin composition occurred over the sampling period and were related to changes in cell concentrations of Alexandrium Halim, Dinophysis Ehrenberg and Pseudo-nitzschia (Hasle) Hasle. Spirolides peaked in late spring and early summer, followed by DA in mid-July. Okadaic acid, DTX1 and PTXs occurred throughout the field season but peaked in late summer. Concentrations of some phycotoxins detected in SPATT samplers deployed within the area where mussels were suspended on lines were lower than in those deployed outside the mussel farm. The SPATT samplers provided a useful tool to detect the presence of phycotoxins and to establish trends in their appearance in the Ship Harbour estuary.

Production of the dinoflagellate Amphidoma languida in a large scale photobioreactor and structure elucidation of its main metabolite AZA-39.

Shellfish contamination with azaspiracids (AZA) is a major and recurrent problem for the Irish shellfish industry. Amphidoma languida, a small thecate dinoflagellate of the family Amphidomataceae, is widely distributed in Irish coastal waters and is one of the identified source species of azaspiracids. Irish and North Sea strains of Am. languida have been found to produce as major metabolites AZA-38 and -39 whose structures have only been provisionally elucidated by mass spectrometry and their toxic potential is currently unknown. In order to provide pure AZA-38 and -39 for subsequent structural and toxicological analyses, we present the first successful large-scale culture of Am. languida. A 180 L in house prototype bioreactor was used for culture growth and harvesting in semi-continuous mode for two months. Two different runs of the photobiorector with different light and pH setting showed the highest toxin yield at higher light intensity and slightly higher pH. AZA-38 and -39 cell quota were measured throughout the complete growth cycle with AZA-39 cell quota increasing in proportion to AZA-38 at late stationary to senescence phase. Over two experiments a total of 700 L of culture was harvested yielding 0.45 mg of pure AZA-39. The structure of AZA-39 was elucidated through NMR data analyses, which led to a revision of the structure proposed previously by mass spectrometry. While the spirotetrahydrofuran/tetrahydrofuran of rings A and B has been confirmed by NMR for AZA-39, a methyl is still present in position C-14 and the carboxylic acid chain is different from the structure proposed initially.

Variation profile of diarrhetic shellfish toxins and diol esters derivatives of Prorocentrum lima during growth by high-resolution mass spectrometry.

Prorocentrum lima is a widely distributed toxigenic benthic dinoflagellate whose production of diarrhetic shellfish toxins threatens the shellfish industry and seafood safety. Current research primarily assesses the difference between free and post-hydrolysis total toxin methods, ignoring the impact of different detection methods on technical accuracy. After removing matrix interference with SPE extraction, a thorough HRMS strategy was created in this study. Alkaline hydrolysis could release the diol esters and played a crucial role in obtaining an accurate assessment of toxin levels, achieving satisfactory recoveries (74.0-147.0%) and repeatability (relative deviation <12.3%). The HRMS approach evaluated toxin profile variation during the growth of three P. lima strains from China. A total of 24 toxin contents varying in composition, content, and a high proportion were detected. The SHG, HN, and 3XS strains had total toxin contents of 23.3 ± 1.74, 19.8 ± 1.25, and 19.5 ± 1.58 pg cell-1, respectively. The diol esters proportion varied among the strains, with SHG having 58.9-69.9, HN having 75.4-86.5, and 3XS having 91.0-91.7%. The variety of toxins produced by distinct P. lima strains highlighted the significance of this method for appropriately measuring the risks connected with DSTs manufacturing. The proposed approach provides a technical basis for gathering comprehensive and accurate data on the potential risks of P. lima DSTs production, with significant implications for ensuring food safety and preventing harmful toxins from spreading in the marine ecosystem.

Ciguatera Fish Poisoning in the Caribbean Sea and Atlantic Ocean: Reconciling the Multiplicity of Ciguatoxins and Analytical Chemistry Approach for Public Health Safety.

Ciguatera is a major circumtropical poisoning caused by the consumption of marine fish and invertebrates contaminated with ciguatoxins (CTXs): neurotoxins produced by endemic and benthic dinoflagellates which are biotransformed in the fish food-web. We provide a history of ciguatera research conducted over the past 70 years on ciguatoxins from the Pacific Ocean (P-CTXs) and Caribbean Sea (C-CTXs) and describe their main chemical, biochemical, and toxicological properties. Currently, there is no official method for the extraction and quantification of ciguatoxins, regardless their origin, mainly due to limited CTX-certified reference materials. In this review, the extraction and purification procedures of C-CTXs are investigated, considering specific objectives such as isolating reference materials, analysing fish toxin profiles, or ensuring food safety control. Certain in vitro assays may provide sufficient sensitivity to detect C-CTXs at sub-ppb levels in fish, but they do not allow for individual identification of CTXs. Recent advances in analysis using liquid chromatography coupled with low- or high-resolution mass spectrometry provide new opportunities to identify known C-CTXs, to gain structural insights into new analogues, and to quantify C-CTXs. Together, these methods reveal that ciguatera arises from a multiplicity of CTXs, although one major form (C-CTX-1) seems to dominate. However, questions arise regarding the abundance and instability of certain C-CTXs, which are further complicated by the wide array of CTX-producing dinoflagellates and fish vectors. Further research is needed to assess the toxic potential of the new C-CTX and their role in ciguatera fish poisoning. With the identification of C-CTXs in the coastal USA and Eastern Atlantic Ocean, the investigation of ciguatera fish poisoning is now a truly global effort.

Structure and toxicity of AZA-59, an azaspiracid shellfish poisoning toxin produced by Azadinium poporum (Dinophyceae).

To date, the putative shellfish toxin azaspiracid 59 (AZA-59) produced by Azadinium poporum (Dinophyceae) has been the only AZA found in isolates from the Pacific Northwest coast of the USA (Northeast Pacific Ocean). Anecdotal reports of sporadic diarrhetic shellfish poisoning-like illness, with the absence of DSP toxin or Vibrio contamination, led to efforts to look for other potential toxins, such as AZAs, in water and shellfish from the region. A. poporum was found in Puget Sound and the outer coast of Washington State, USA, and a novel AZA (putative AZA-59) was detected in low quantities in SPATT resins and shellfish. Here, an A. poporum strain from Puget Sound was mass-cultured and AZA-59 was subsequently purified and structurally characterized. In vitro cytotoxicity of AZA-59 towards Jurkat T lymphocytes and acute intraperitoneal toxicity in mice in comparison to AZA-1 allowed the derivation of a provisional toxicity equivalency factor of 0.8 for AZA-59. Quantification of AZA-59 using ELISA and LC-MS/MS yielded reasonable quantitative results when AZA-1 was used as an external reference standard. This study assesses the toxic potency of AZA-59 and will inform guidelines for its potential monitoring in case of increasing toxin levels in edible shellfish.

Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis.

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways.

Discovering a New Okadaic Acid Derivative, a Potent HIV Latency Reversing Agent from Prorocentrum lima PL11: Isolation, Structural Modification, and Mechanistic Study.

Marine toxins (MTs) are a group of structurally complex natural products with unique toxicological and pharmacological activities. In the present study, two common shellfish toxins, okadaic acid (OA) (1) and OA methyl ester (2), were isolated from the cultured microalgae strain Prorocentrum lima PL11. OA can significantly activate the latent HIV but has severe toxicity. To obtain more tolerable and potent latency reversing agents (LRAs), we conducted the structural modification of OA by esterification, yielding one known compound (3) and four new derivatives (4-7). Flow cytometry-based HIV latency reversal activity screening showed that compound 7 possessed a stronger activity (EC50 = 46 ± 13.5 nM) but was less cytotoxic than OA. The preliminary structure-activity relationships (SARs) indicated that the carboxyl group in OA was essential for activity, while the esterification of carboxyl or free hydroxyls were beneficial for reducing cytotoxicity. A mechanistic study revealed that compound 7 promotes the dissociation of P-TEFb from the 7SK snRNP complex to reactivate latent HIV-1. Our study provides significant clues for OA-based HIV LRA discovery.

Multiply charged ion profiles in the UHPLC-HRMS analysis of palytoxin analogues from Ostreopsis cf. ovata blooms.

Analogues of palytoxin (PLTX), one of the most potent marine biotoxins, are produced by some species of the marine dinoflagellates of the genus Ostreopsis. The proliferation of these species in different coastal zones represents a potential threat of seafood poisoning in humans because the produced toxins can be transferred through marine food webs. Thus, the determination of the concentration of PLTX analogues (ovatoxins-OVTXs, ostreocins-OSTs and isobaric PLTX) in different matrices (seawater, marine fauna, etc.) is necessary to protect human health. This study is addressed to overcome some of the challenges that the chemical complexity of these molecules poses to their quantification by ultra-high-performance liquid chromatography high-resolution mass spectrometry-based techniques (UHPLC-HRMS). In particular, the mass spectra of the palytoxin analogues show the presence of a large number of ions (including mono- and multiply charged ions) whose nature, relative abundances and behavior can lead to quantitation errors if the correct ions are not selected. In this work, the variability of the PLTX and OVTX profiles under different instrument conditions, including the use of diverse electrospray generation sources and different quantitation methods, is studied. Moreover, the extraction protocol in seawater containing Ostreopsis sp. ovata cells is also evaluated. The use of a heated electrospray operating at 350 °C and a quantitative method including ions from different multiply charged species provides a more robust and reliable method for overcoming the problems due to the variability in the toxin's mass spectrum profile. A single MeOH : H2O (80 : 20, v/v) extraction is proposed as the best and reliable procedure. The overall method proposed was applied to quantify OVTXs (-a to -g) and iso-PLTX along the 2019 Ostreopsis cf. ovata bloom. The cells contained a total toxin concentration of up to 20.39 pg per cell.

Regional comparison on ciguatoxicity, hemolytic activity, and toxin profile of the dinoflagellate Gambierdiscus from Kiribati and Malaysia.

The dinoflagellates Gambierdiscus and Fukuyoa can produce Ciguatoxins (CTXs) and Maitotoxins (MTXs) that lead to ciguatera poisoning (CP). The CP hotspots, however, do not directly relate to the occurrence of the ciguatoxic Gambierdiscus and Fukuyoa. Species-wide investigations often showed no association between CTX level and the molecular identity of the dinoflagellates. In the Pacific region, Kiribati is known as a CP hotspot, while Malaysia has only three CP outbreaks reported thus far. Although ciguatoxic strains of Gambierdiscus were isolated from both Kiribati and Malaysia, no solid evidence on the contribution of ciguatoxic strains to the incidence of CP outbreak was recorded. The present study aims to investigate the regional differences in CP risks through region-specific toxicological assessment of Gambierdiscus and Fukuyoa. A total of 19 strains of Gambierdiscus and a strain of Fukuyoa were analyzed by cytotoxicity assay of the neuro-2a cell line, hemolytic assay of fish erythrocytes, and high-resolution mass spectrometry. Gambierdiscus from both Kiribati and Malaysia showed detectable ciguatoxicity; however, the Kiribati strains were more hemolytic. Putative 44-methylgambierone was identified as part of the contributors to the hemolytic activity, and other unknown hydrophilic toxins produced can be potentially linked to higher CP incidence in Kiribati.